The peptidoglycan (PG) layer stabilizes the bacterial cell envelope to maintain the integrity and shape of the cell. Penicillin-binding proteins (PBPs) synthesize essential 4–3 cross-links in PG and are inhibited by β-lactam antibiotics. Some clinical isolates and laboratory strains of Enterococcus faecium and Escherichia coli achieve high-level β-lactam resistance by utilizing β-lactam–insensitive LD-transpeptidases (LDTs) to produce exclusively 3–3 cross-links in PG, bypassing the PBPs. In E. coli, other LDTs covalently attach the lipoprotein Lpp to PG to stabilize the envelope and maintain the permeability barrier function of the outermembrane. Here we show that subminimal inhibitory concentration of copper chloride sensitizes E. coli cells to sodium dodecyl sulfate and impair survival upon LPS transport stress, indicating reduced cell envelope robustness. Cells grown in the presence of copper chloride lacked 3–3 cross-links in PG and displayed reduced covalent attachment of Braun’s lipoprotein and reduced incorporation of a fluorescent d-amino acid, suggesting inhibition of LDTs. Copper dramatically decreased the minimal inhibitory concentration of ampicillin in E. coli and E. faecium strains with a resistance mechanism relying on LDTs and inhibited purified LDTs at submillimolar concentrations. Hence, our work reveals how copper affects bacterial cell envelope stability and counteracts LDT-mediated β-lactam resistance.

difficile-associated diseases. 52The structure of cell wall peptidoglycan is unusual in C. difficile since the majority

β-lactam antibiotics act as suicide substrates of transpeptidases responsible for the last cross-linking step of peptidoglycan synthesis in the bacterial cell wall. Nucleophilic attack of the β-lactam carbonyl by the catalytic residue (Ser or Cys) of transpeptidases results in the opening of the β-lactam ring and in the formation of a stable acyl-enzyme. The acylation reaction is considered as irreversible due to the strain of the β-lactam ring. In contradiction with this widely accepted but poorly demonstrated premise, we show here that the acylation of the L,D-transpeptidase Ldtfm from Enterococcus faecium by the β-lactam nitrocefin is reversible, leading to limited antibacterial activity. Experimentally, two independent methods based on spectrophotometry and mass spectrometry provided evidence that recyclization of the β-lactam ring within the active site of Ldtfm regenerates native nitrocefin. Ring strain is therefore not sufficient to account for irreversible acylation of peptidoglycan transpeptidases observed for most β-lactam antibiotics.

There is a renewed interest for β-lactams for treating infections due to Mycobacterium tuberculosis and M. abscessus because their β-lactamases are inhibited by classical (clavulanate) or new generation (avibactam) inhibitors, respectively. Here, access to an azido derivative of the diazabicyclooctane (DBO) scaffold of avibactam for functionalization by the Huisgen-Sharpless cycloaddition reaction is reported. The amoxicillin-DBO combinations were active, indicating that the triazole ring is compatible with drug penetration (minimal inhibitory concentration of 16 μg mL for both species). Mechanistically, β-lactamase inhibition was not sufficient to account for the potentiation of amoxicillin by DBOs. Thus, the latter compounds were investigated as inhibitors of l,d-transpeptidases (Ldts), which are the main peptidoglycan polymerases in mycobacteria. The DBOs acted as slow-binding inhibitors of Ldts by S-carbamoylation indicating that optimization of DBOs for Ldt inhibition is an attractive strategy to obtain drugs selectively active on mycobacteria.

Second-generation-lactamase inhibitors containing a dia abic clooctane (DBO) scaffold restore the activit of-lactams against pathogenic bacteria, including those producing class A, C, and D en mes that are not susceptible to first-generation inhibitors containing a-lactam ring. Here, e report optimi ation of a s nthetic route to access tria olecontaining DBOs and biological evaluation of a series of 17 compounds for inhibition of five-lactamases representative of en mes found in pathogenic Gram-negative bacteria. A strong correlation (Spearman coefficient of 0.87; = 4.7 10-21) as observed bet een the inhibition efficac of purified-lactamases and the potentiation of-lactam antibacterial activit indicating that DBO functionali ation did not impair penetration. In comparison to reference DBOs, avibactam and relebactam, our compounds displa ed reduced efficac likel due to the absence of h drogen bonding ith a conserved asparagine residue at position 132. This as partiall compensated b additional interactions involving certain tria ole substituents.

As β-lactams are reconsidered for the treatment of tuberculosis (TB), their targets are assumed to be peptidoglycan transpeptidases, as verified by adduct formation and kinetic inhibition of Mycobacterium tuberculosis (Mtb) transpeptidases by carbapenems active against replicating Mtb. Here, we investigated the targets of recently described cephalosporins that are selectively active against non-replicating (NR) Mtb. NR-active cephalosporins failed to inhibit recombinant Mtb transpeptidases. Accordingly, we used alkyne analogs of NR-active cephalosporins to pull down potential targets through unbiased activity-based protein profiling and identified over 30 protein binders. None was a transpeptidase. Several of the target candidates are plausibly related to Mtb's survival in an NR state. However, biochemical tests and studies of loss of function mutants did not identify a unique target that accounts for the bactericidal activity of these beta-lactams against NR Mtb. Instead, NR-active cephalosporins appear to kill Mtb by collective action on multiple targets. These results highlight the ability of these β-lactams to target diverse classes of proteins.

The Enterococcus faecium L,D-transpeptidase (Ldt fm ) mediates resistance to most ␤-lactam antibiotics in this bacterium by replacing classical peptidoglycan polymerases. The catalytic Cys of Ldt fm is rapidly acylated by ␤-lactams belonging to the carbapenem class but not by penams or cephems. We previously reported quantum calculations and kinetic analyses for Ldt fm and showed that the inactivation profile is not determined by differences in drug binding (K D [equilibrium dissociation constant] values in the 50 to 80 mM range). In this study, we analyzed the reaction of a Cys sulfhydryl with various ␤-lactams in the absence of the enzyme environment in order to compare the intrinsic reactivity of drugs belonging to the penam, cephem, and carbapenem classes. For this purpose, we synthesized cyclic Cys-Asn (cCys-Asn) to generate a soluble molecule with a sulfhydryl closely mimicking a cysteine in a polypeptide chain, thereby avoiding free reactive amino and carboxyl groups. Computational studies identified a thermodynamically favored pathway involving a concerted rupture of the ␤-lactam amide bond and formation of an amine anion. Energy barriers indicated that the drug reactivity was the highest for nonmethylated carbapenems, intermediate for methylated carbapenems and cephems, and the lowest for penams. Electron-withdrawing groups were key reactivity determinants by enabling delocalization of the negative charge of the amine anion. Acylation rates of cCys-Asn determined by spectrophotometry revealed the same order in the reactivity of ␤-lactams. We concluded that the rate of Ldt fm acylation is largely determined by the ␤-lactam reactivity with one exception, as the enzyme catalytic pocket fully compensated for the detrimental effect of carbapenem methylation.

In most bacteria, β-lactam antibiotics inhibit the last cross-linking step of peptidoglycan synthesis by acylation of the active-site Ser of D,D-transpeptidases belonging to the penicillin-binding protein (PBP) family. In mycobacteria, cross-linking is mainly ensured by L,D-transpeptidases (LDTs), which are promising targets for the development of β-lactam-based therapies for multidrug-resistant tuberculosis. For this purpose, fluorescence spectroscopy is used to investigate the efficacy of LDT inactivation by β-lactams but the basis for fluorescence quenching during enzyme acylation remains unknown. In contrast to what has been reported for PBPs, we show here using a model L,Dtranspeptidase (Ldtfm) that fluorescence quenching of Trp residues does not depend upon direct hydrophobic interaction between Trp residues and β-lactams. Rather, Trp fluorescence was quenched by the drug covalently bound to the active-site Cys residue of Ldtfm. Fluorescence quenching was not quantitatively determined by the size of the drug and was not specific of the thioester link connecting the β-lactam carbonyl to the catalytic Cys as quenching was also observed for acylation of the active-site Ser of β-lactamase BlaC from M. tuberculosis. Fluorescence quenching was extensive for reaction intermediates containing an amine anion and for acylenzymes containing an imine stabilized by mesomeric effect, but not for acylenzymes containing a protonated β-lactam nitrogen. Together, these results indicate that the extent of fluorescence quenching is determined by the status of the β-lactam nitrogen. Thus, fluorescence kinetics can provide information not only on the efficacy of enzyme inactivation but also on the structure of the covalent adducts responsible for enzyme inactivation.Peptidoglycan is an essential constituent of bacterial cell walls since it prevents cell swelling and lysis by mechanically sustaining the osmotic pressure of the cytoplasm 1 . This protective function depends upon synthesis and maintenance during the entire cell cycle of the net-like peptidoglycan macromolecule, which completely surrounds the bacterial cell. Peptidoglycan is made of glycan strands cross-linked by short peptide stems. In most bacteria, the cross-linking step is performed by D,Dtranspeptidases, which are the essential targets of β-lactam antibiotics and are often referred to as penicillin-binding proteins (PBPs) 2 . In mycobacteria [3][4] and in Clostridium difficile [5][6] , the cross-links found are mainly (70% to 80%) formed by a second class of enzymes, the L,D-transpeptidases (LDTs). Since LDTs are not inhibited by β-lactams belonging to the penam class, such as ampicillin, 7-8 , these enzymes are responsible for high-level resistance to these drugs in mutants of Enterococcus faecium 9-10 and Escherichia coli 11 selected in laboratory conditions.PBPs and LDTs are structurally unrelated [12][13][14][15] and proceed through different catalytic mechanisms for activation of Ser and Cys nucleophiles 16 , which are part of Lys-Ser 17 and Cys-His-Asp 18...